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PloS One 2015The genetic diversity and population structure of Vibrio vulnificus isolates from Korea and Taiwan were investigated using PCR-based assays targeting putative...
The genetic diversity and population structure of Vibrio vulnificus isolates from Korea and Taiwan were investigated using PCR-based assays targeting putative virulence-related genes and multilocus sequence typing (MLST). BOX-PCR genomic fingerprinting identified 52 unique genotypes in 84 environmental and clinical V. vulnificus isolates. The majority (> 50%) of strains had pathogenic genotypes for all loci tested; moreover, many environmental strains had pathogenic genotypes. Although significant (p < 0.05) inter-relationships among the genotypes were observed, the association between genotype and strain source (environmental or clinical) was not significant, indicating that genotypic characteristics alone are not sufficient to predict the isolation source or the virulence of a given V. vulnificus strain and vice versa. MLST revealed 23-35 allelic types per locus analyzed, resulting in a total of 44 unique sequence types (STs). Two major monophyletic groups (lineages A and B) corresponding to the two known lineages of V. vulnificus were observed; lineage A had six STs that were exclusively environmental, whereas lineage B had STs from both environmental and clinical sources. Pathogenic and nonpathogenic genotypes predominated in MLST lineages B and A, respectively. In addition, V. vulnificus was shown to be in linkage disequilibrium (p < 0.05), although two different recombination tests (PHI and Sawyer's tests) detected significant evidence of recombination. Tajima's D test also indicated that V. vulnificus might be comprised of recently sub-divided lineages. These results suggested that the two lineages revealed by MLST correspond to two distinct ecotypes of V. vulnificus.
Topics: DNA Fingerprinting; Genetic Variation; Genetics, Population; Humans; Korea; Linkage Disequilibrium; Multilocus Sequence Typing; Phylogeny; RNA, Ribosomal; Republic of Korea; Seafood; Sequence Analysis, DNA; Taiwan; Vibrio Infections; Vibrio vulnificus
PubMed: 26599487
DOI: 10.1371/journal.pone.0142657 -
Biological & Pharmaceutical Bulletin 2022Vibrio vulnificus is a Gram-negative estuarine bacterium that causes infection in immuno-compromised patients, eels, and shrimp. V. vulnificus NCIMB2137, a...
Vibrio vulnificus is a Gram-negative estuarine bacterium that causes infection in immuno-compromised patients, eels, and shrimp. V. vulnificus NCIMB2137, a metalloprotease-negative strain isolated from a diseased eel, produces a 45-kDa chymotrypsin-like alkaline serine protease known as VvsA. The gene encoding vvsA also includes another gene, vvsB with an unknown function; however, it is assumed to be an essential molecular chaperone for the maturation of VvsA. In the present study, we used an in vitro cell-free translation system to examine the maturation pathway of VvsA. We individually expressed the vvsA and vvsB genes and detected their mRNAs. However, the sample produced from vvsA did not exhibit protease activity. A sodium dodecyl sulfate (SDS) analysis detected the VvsB protein, but not the VvsA protein. A Western blotting analysis using a histidine (His)-tag at the amino terminus of proteins also showed no protein production by vvsA. These results suggested the translation, but not the transcription of vvsA. Factors derived from Escherichia coli were used in the in vitro cell-free translation system employed in the present study. The operon of the serine protease gene containing vvsA and vvsB was expressed in E. coli. Although serine proteases were produced, they were cleaved at different sites and no active mature forms were detected. These results indicate that the operon encoding vvsA and vvsB is a gene constructed to be specifically expressed in V. vulnificus.
Topics: Humans; Vibrio vulnificus; Serine Proteases; Escherichia coli; Serine Endopeptidases
PubMed: 36328494
DOI: 10.1248/bpb.b22-00106 -
The Journal of Veterinary Medical... Jul 2015Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged...
Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.
Topics: Animals; DNA Probes; Female; Genes, Bacterial; Mice; Mice, Inbred ICR; Mutagenesis, Insertional; Vibrio vulnificus; Virulence Factors
PubMed: 25755021
DOI: 10.1292/jvms.14-0655 -
PloS One 2014Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question...
BACKGROUND
Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species.
RESULTS
In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5'-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218.
CONCLUSION
We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.
Topics: Bacteriophages; Base Sequence; Genome, Viral; Humans; Lysogeny; Microscopy, Electron, Transmission; Mitomycin; Molecular Sequence Data; Open Reading Frames; Prophages; Transcription, Genetic; Vibrio; Vibrio cholerae; Vibrio vulnificus; Virulence
PubMed: 24732980
DOI: 10.1371/journal.pone.0094707 -
Infection and Immunity Apr 2008Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus...
Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus that exhibited less cytotoxicity to INT-407 human intestinal epithelial cells was screened from a library of mutants constructed by random transposon mutagenesis. A transposon-tagging method was used to identify and clone an open reading frame encoding an RTX toxin secretion ATP binding protein, RtxE, from V. vulnificus. The deduced amino acid sequence of RtxE from V. vulnificus was 91% identical to that reported from Vibrio cholerae. Functions of the rtxE gene in virulence were assessed by constructing an isogenic mutant whose rtxE gene was inactivated by allelic exchanges and by evaluating the differences between its virulence phenotype and that of the wild type in vitro and in mice. The disruption of rtxE blocked secretion of RtxA to the cell exterior and resulted in a significant reduction in cytotoxic activity against epithelial cells in vitro. Also, the intraperitoneal 50% lethal dose of the rtxE mutant was 10(4) to 10(5) times higher than that of the parental wild type, indicating that RtxE is essential for the virulence of V. vulnificus. Furthermore, the present study demonstrated that the rtxBDE genes are transcribed as one transcriptional unit under the control of a single promoter, P(rtxBDE). The activity of V. vulnificus P(rtxBDE) is induced by exposure to INT-407 cells, and the induction requires direct contact of the bacteria with the host cells.
Topics: Animals; Cell Line; Epithelial Cells; Gene Expression Regulation, Bacterial; Humans; Iron, Dietary; Mice; Mutation; Transcription, Genetic; Vibrio Infections; Vibrio vulnificus; Virulence; Virulence Factors
PubMed: 18250174
DOI: 10.1128/IAI.01503-07 -
Emerging Microbes & Infections 2019Cytoskeletal rearrangement and acute cytotoxicity occur in infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for...
Cytoskeletal rearrangement and acute cytotoxicity occur in infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491-1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA1 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA1 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.
Topics: Amino Acid Motifs; Bacterial Toxins; Cell Death; Cytoskeleton; Filamins; HeLa Cells; Host-Pathogen Interactions; Humans; Mitogen-Activated Protein Kinase Kinases; Protein Binding; Vibrio Infections; Vibrio vulnificus; p21-Activated Kinases
PubMed: 31237474
DOI: 10.1080/22221751.2019.1632153 -
Applied and Environmental Microbiology Dec 2014Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters,...
Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)-real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh(+) and/or trh(+)) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and -0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.
Topics: Animals; Bivalvia; Crassostrea; Food Contamination; Mercenaria; New York; Ostreidae; Vibrio cholerae; Vibrio parahaemolyticus; Vibrio vulnificus
PubMed: 25281373
DOI: 10.1128/AEM.02820-14 -
Applied and Environmental Microbiology Apr 2015The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently...
The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.
Topics: Animals; Aquatic Organisms; Estuaries; Florida; Geologic Sediments; Gulf of Mexico; Ostreidae; Plants; Real-Time Polymerase Chain Reaction; Salinity; Seawater; Temperature; Vibrio vulnificus
PubMed: 25636843
DOI: 10.1128/AEM.03243-14 -
The Journal of Biological Chemistry Apr 2020For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner....
For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen produces a cytolysin/hemolysin protein encoded by the operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the promoter P , which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates transcription by directly binding upstream of P , whereas the histone-like nucleoid-structuring protein (H-NS) represses by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the P regulatory region, resulting in the release of H-NS repression and induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of P provides with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of transcription, thereby enabling successful host infection.
Topics: Animals; Bacterial Proteins; Bacterial Toxins; Cells, Cultured; Gene Expression Regulation, Bacterial; Iron; Iron Deficiencies; Mice; Nitrogen; Operon; Promoter Regions, Genetic; RAW 264.7 Cells; Stress, Physiological; Transcription Factors; Vibrio vulnificus
PubMed: 32169898
DOI: 10.1074/jbc.RA120.012724 -
Journal of Food Protection Dec 2013Vibrio vulnificus is a gram-negative bacterium that occurs naturally in estuarine and marine water and is associated with wound infections or septicemia related to the...
Vibrio vulnificus is a gram-negative bacterium that occurs naturally in estuarine and marine water and is associated with wound infections or septicemia related to the consumption of raw shellfish in humans. The molecular characteristics and antibiotic susceptibilities of V. vulnificus strains in shrimps from retail markets in Hangzhou, People's Republic of China, were investigated in this study. Thirty-three samples were positive for V. vulnificus in 78 shrimp samples which were collected from 15 retail markets between July and August 2012; the most-probable-number values ranged from 3 to 1,600 g(-1) in these positive samples, with a median most-probable-number value of 72 g(-1). Twenty-five biotype 1 strains and eight biotype 2 strains were identified by biochemical tests, and all strains could be definitively genotyped. By 16S rRNA genotyping, 21.2% (7 of 33) were classified as genotype A, 63.6% (21 of 33) as genotype B, and 15.2% (5 of 33) as genotype AB, while by virulence-correlated gene (vcg) typing, 21.2% (7 of 33) were characterized as genotype E and 78.8% (26 of 33) were genotype C. More than 50% of those isolates were identified as the potentially virulent type vcg type C-16S rRNA B (CB). The antibiotic susceptibilities of the V. vulnificus strains to 21 antimicrobial agents were tested as well. Some strains showed resistance or intermediate resistance to cefepime (3.03%), tetracycline (6.06%), aztreonam (24.24%), streptomycin (45.45%), gentamicin (93.94%), tobramycin (100%), and cefazolin (100%). Multiple-locus variable-number tandem repeat-based fingerprinting analysis (MLVA) was successfully applied to these 33 isolates and yielded 30 patterns that clustered into two MLVA groups; with a calculated Simpson's index of diversity of 0.994, this revealed that MLVA had great discriminating power for V. vulnificus. To minimize the potential risk of V. vulnificus infections due to the consumption of raw shrimp, it is necessary to monitor the hygiene status of seafood.
Topics: Animals; Anti-Bacterial Agents; China; Colony Count, Microbial; Drug Resistance, Bacterial; Food Contamination; Genotype; Humans; Penaeidae; RNA, Ribosomal, 16S; Shellfish; Vibrio vulnificus; Virulence
PubMed: 24290683
DOI: 10.4315/0362-028X.JFP-13-161